As espécies do gênero Viguiera Kunth, recentemente transferidas para Aldama La Llave (Asteraceae, Heliantheae), ainda possuem problemas de delimitação taxonômica. Tal gênero possui 35 espécies distribuídas por todo o território brasileiro, principalmente no cerrado. Análises químicas têm demonstrado seu potencial em auxiliar na resolução de problemas em vários níveis taxonômicos com base em grupos químicos de metabólitos secundários. Além disso, análises fitoquímicas tem revelado que determinadas substâncias de Aldama possuem potencial para serem estudadas tendo vista a investigação de mecanismos moleculares anti-inflamatórios. Desta forma, foi proposto neste trabalho realizar a abordagem da metabolômica não-direcionada auxiliada por quimiometria, visando-se fornecer dados químicos tanto para contribuições quimiotaxonômicas quanto para encontrar substâncias bioativas. Por meio de tal abordagem, análises multivariadas não supervisionadas (PCA e HCA), bem como supervisionadas (OPLS-DA), com dados provenientes de UHPLC-DAD-Orbitrap, demonstraram que o gênero Aldama é quimioconsistente e, deste modo, determinadas substâncias químicas discriminantes foram sugeridas. Além disso, espécies que apresentam problemas taxonômicos tiveram os seus posicionamentos infragenéricos explicado do ponto de vista quimiotaxonômico. Quanto às análises para encontrar substâncias bioativas (fitoquímica direcionada), os alvos inflamatórios pesquisados foram as enzimas pró-inflamatórias COX-1 e 5-LOX, com as quais se realizou ensaios in vitro verificando-se a atividade positiva de extratos de espécies de Aldama. A. trichophylla foi a espécie selecionada para realizar a fitoquímica direcionada, uma vez que apresentou inibição dupla contra as enzimas e por se tratar de uma espécie muito pouco estudada do ponto de vista fitoquímico. Os melhores modelos de inibição de COX-1 e 5-LOX criados com dados químicos de Aldama foram selecionados a partir de diversas combinações de algoritmos, softwares de processamento (MZmine, MetAlign e MSClust) e técnicas-hifenadas, tais como UHPLC-Orbitrap e HPLC-TOF. Deste modo, determinados picos de íons foram apontados pela quimiometria como sendo discriminantes para a atividade de inibição dupla. Tais picos foram desreplicados com base em seus perfis de UV e padrões de fragmentação via HCD-Orbitrap e Ion-Trap. Deste modo, foi possível desreplicar três prováveis substâncias químicas inibidoras de COX-1 e 5-LOX: kaempferol-3-O-glucuronideo, quercetina-3-O-metil-7-glucuronideo e kaempferol-3-O-(6\"-malonil-glucosideo). Por fim, foi realizada a fitoquímica direcionada. O fracionamento cromatográfico permitiu isolar tais substâncias e uma análise preliminar de RMN 1H foi realizada com o intuito de realizar a identificação estrutural. Tais substâncias terão a suas atividades confirmadas na inibição das duas enzimas em um futuro próximo. / The species of the genus Viguiera Kunth, recently transferred to the genus Aldama La Llave (Asteraceae, Heliantheae), still show problems of taxonomic boundaries. This genus has 35 species distributed throughout the Brazilian territory, mostly in the cerrado. Chemical analyses has demonstrated its potential to help in the solution of problems at various taxonomic levels based on chemical groups of secondary metabolites. Furthermore, phytochemical analyses have shown that certain compounds from Aldama have the potential to be studied aiming to investigate anti-inflammatory molecular mechanisms. Thus, in this work it was proposed to carry out the untargeted metabolomic approach aided by chemometrics, aiming to provide chemical data either for chemotaxonomic contributions and to find bioactive compounds. Through this approach, both unsupervised (PCA and HCA) and supervised multivariate analysis (OPLS-DA) combined with data from UHPLC-DAD-Orbitrap analyses showed that the genus Aldama is \"chemoconsistent\" and thus certain discriminant compounds were suggested. Additionally, some species with taxonomic problems had their infrageneric positioning explained from the chemotaxonomic point of view of. With regards to the analyses carried out to find bioactive compounds (targeted phytochemistry), the inflammatory targets investigated in this study were the COX-1 and LOX-5 pro-inflammatory enzymes with which in vitro assays were made and positive activity of extracts from species of Aldama was observed. A. trichophylla was the selected species for the targeted phytochemistry because it showed dual inhibition activity against both enzymes and it is still little investigated from the chemical point of view. The best models for inhibition of COX-1 and 5-LOX obtained with chemical data of Aldama were selected by means of various combinations of algorithms, processing software (MZmine, MetAlign and MSClust) and hyphenated techniques, such as HPLC-TOF and UHPLC-Orbitrap. Thus, certain peak ions were appointed by chemometrics as discriminant for the dual inhibition activity. These peaks were dereplicated based on their UV profiles and fragmentation patterns via HCD-Orbitrap and Ion-Trap. Thus, it was possible to dereplicate three probable chemical inhibitors of COX-1 and 5-LOX: kaempferol-3-O-glucuronide, quercetin-3-O-methyl-7-glucuronide and kaempferol-3-O-(6\"-malonyl-glucoside). Finally, the targeted phytochemistry was carried out. The chromatographic fractionation allowed us to isolate these three compounds and a preliminary 1H NMR analysis was performed in order to conclude their structural identification. In the near future, these substances will have their activity evaluated by the inhibition of the two enzymes.

No description available.

No description available.

Le travail de thèse décrit dans ce manuscrit porte sur l’étude de la présence de biocides désinfectants dans les produits laitiers et sur l’impact des procédés de transformation du lait sur l’apparition de produits néoformés. Les biocides désinfectants sont des composés chimiques utilisés quotidiennement en industrie laitière lors des procédures de nettoyage-désinfection (ND) des surfaces en contact avec les aliments. Les ammoniums quaternaires de type chlorure de Benzalkonium (BACs) et chlorure de dialkyldimethylammonium (DDACs), et l’Aminopropyldodécylpropane diamine sont les désinfectants les plus largement utilisés en industrie laitière. Ces biocides peuvent entrainer des résidus sur les surfaces alimentaires, ce qui présente un risque pour la santé du consommateur. Dans le but de mesurer l’exposition des consommateurs, deux méthodes analytiques fiables ont été développées pour l'analyse de ces substances dans les produits laitiers impliquant la chromatographie liquide couplée à la spectrométrie de masse en tandem. Le lait cru de vache, la poudre de lait entier, les fromages à pâte pressée cuite et les fromages fondus ont été sélectionnés pour représenter la diversité des produits laitiers. L'évaluation des performances de chacune des méthodes a été réalisée par une approche globale basée sur le profil d'exactitude. Pour la plupart des composés et des matrices étudiés, les méthodes d'analyse ont été validées sur l’intervalle de dosage de 5 à 150 μg / kg. Pour répondre au questionnement de l'impact des procédés de transformation du lait sur les résidus de biocides désinfectants, l'évolution des teneurs en composés et leur devenir dans les différentes matrices issues du lait ont été étudiées. Pour cela, deux études de faisabilité mettant en oeuvre des comparaisons d'empreintes chimiques globales, acquises par spectrométrie de masse à haute résolution, de fromages fondus et de fromages à pâte dure (contaminés vs témoins) ont été réalisées. Ces études ont permis de détecter 4 ions discriminants liés à la présence de biocides dans le fromage fondu. Leur identification reste à réaliser. Tout ce travail a été réalisé à des fins de sécurité alimentaire. La première partie est à l'élaboration de méthodes analytiques ciblées pour les résidus de biocides dans le lait et les produits laitiers permettant ainsi de mesurer les résidus de biocides sur les denrées alimentaires. Ces mesures sont nécessaires pour effectuer une analyse du risque liée à ces résidus. La deuxième partie est en relation avec la question du comportement des résidus de biocides lors de la transformation du lait présentant la stratégie, les résultats que nous pourrions obtenir et la perspective de travaux futurs. / The thesis work focuses on the presence of disinfectants biocides in dairy products and on the impact of milk processing on the possible appearance of process-induced food contaminants related to the exposition of milk with biocides. Disinfectants biocides are chemicals daily used in the dairy industry in cleaning-disinfection (CD) processes of food contact surfaces. Quaternary ammoniums as benzalkonium chloride (BACs) and dialkyldimethylammonium chloride (DDACs), and the Aminopropyldodecylpropane diamine are the most widely used disinfectants in dairy industry. These biocides can lead residues on the surfaces of food contact materials therefore present a health risk for the consumer. With aim of measuring consumers exposure, two reliable analytical methods have been developed for the analysis of these substances in dairy products involving liquid chromatography hyphenated with tandem mass spectrometry. Raw cow's milk, whole milk powder, hard pressed and processed cheeses were selected as representing the diversity of dairy products. The evaluation of the performances of each of the methods was carried out by the global approach based on the accuracy profile. For most of compounds and matrices studied, analytical methods were validated within the range of 5 to 150 μg/kg. To answer to the questioning of the impact of milk processes on biocides disinfectants residues, the evolution of compounds contents and their fate in the various matrices resulting from the milk were studied. For this, two proof-of-concept studies implementing global chemical fingerprint comparisons, acquired by High Resolution Mass Spectrometry, of processed cheese and hard pressed cheese (contaminated vs control) samples were carried out. These studies allowed to detect 4 discriminant ions linked to the presence of biocides in processed cheese. Their identification remains to be done. Whole this work is related for food safety purposes. The first part was linked to elaborate targeted analytical methods for biocides residues in milk and milk products thus allowing the measurement of biocides residues on food. These measurements are necessary for the risk analysis linked to these residues. The second part is in relation with the question of the behavior of biocides residues within milk processing presenting the strategy, the results we could obtain and the perspective for future works.

No description available.

No description available.

Plants can produce a wide variety of volatile organic compounds (VOCs), which are mediators of plant responses against stresses. Abiotic and biotic factors can modulate VOC emission dynamics from plants, suggesting a key role of these compounds in plant defense processes. Although grapevine VOCs responsible for wine aroma and plant-insect communications are well characterized, the functional properties of VOCs produced in response to phytopathogens were less studied. The aims of this work were i) to summarize the information available in the literature on emission patterns and possible biological functions of VOCs produced by grapevine plants in response to abiotic and biotic factors, ii) to investigate the defense mechanisms activated by volatile linalool in grapevine leaves against downy mildew caused by Plasmopara viticola, and iii) to identify metabolic changes associated with VOC-induced resistance activated by 2-phenylethanol and β-cyclocitral against downy mildew. The first chapter includes an introduction to VOCs in plant communication and analytical methods for metabolic detections, while the second chapter defines the aims of the project. In the third chapter, the literature on grapevine VOCs was reviewed and volatile molecules emitted in response to the exogenous stimuli were discussed. The key findings of this chapter are that specific VOC emission patterns can be stimulated by phytopathogens, resistance inducers, beneficial microorganisms, and abiotic factors, suggesting the precise involvement of airborne signals in grapevine responses to abiotic and biotic stresses. In particular, VOCs with inhibitory activities against grapevine pathogens are known to be emitted mainly by resistant grapevine genotypes upon pathogen inoculation, and some of these VOCs were proposed as biomarkers of grapevine resistance traits. In the fourth chapter, defense responses activated by volatile linalool in grapevine leaves were analyzed. Linalool treatment reduced downy mildew severity on leaf disks of susceptible grapevines (cultivar Pinot noir) and stimulated callose deposition at the sites of P. viticola infection. Moreover, the upregulation of defense-related genes was found in linalool-treated leaf disks, indicating the activation of grapevine defense mechanisms related to salicylic acid and jasmonic acid pathways. By ultra-high pressure liquid chromatography-electrospray ionization-high-resolution quadrupole time of flight-mass spectrometry it was demonstrated that linalool treatment caused changes in the leaf metabolome of mock-inoculated and P. viticola-inoculated samples at one and six days post inoculation (dpi). In the fifth chapter, a deep metabolomic analysis of VOC-induced grapevine resistance was carried out by ultra-high pressure liquid chromatography-heated electrospray ionization-high-resolution Orbitrap-mass spectrometry, which allowed accurate mass detection, fragment and isotope pattern matching, and metabolite annotation. In this study, 2-phenylethanol and β-cyclocitral treatment reduced downy mildew severity on leaf disks of susceptible grapevines (cultivar Pinot noir) causing complex changes in the leaf metabolome of mock-inoculated and P. viticola-inoculated samples at 1 dpi and 6 dpi. Metabolic features with significant increases in abundance in 2-phenylethanol- and β-cyclocitral-treated samples mainly belonged to putative carbohydrates and conjugates, carboxylic acids and derivates, carbonyl compounds, phenylpropanoids, and terpenoids, including molecules possibly associated with plant defense against pathogens. Thus, the chemical classification of annotated compounds revealed that VOCs treatments can activate specific metabolic responses in grapevine leaves that include the accumulation of defense-related compounds. These results demonstrated that linalool, 2-phenylethanol, and β-cyclocitral induce grapevine resistance against downy mildew, acting as volatile signaling molecules for plant resistance induction. Although further studies on formulation and efficacy under controlled and field conditions are required, these VOCs have great potential for the further development of sustainable products for downy mildew control.

Grâce à la spectrométrie de masse de type MALDI-TOF, l'identification des bactéries est maintenant possible en quelques minutes. Mais le taux de mortalité des patients augmente lorsqu'une antibiothérapie inappropriée est utilisée et les instruments MALDI-TOF ne sont pas capables d'analyser rapidement et exhaustivement la résistance bactérienne. Actuellement, 6 à 24 heures sont nécessaires pour déterminer le phénotype de résistance. En couplant une chromatographie liquide et un spectromètre de masse à ionisation électrospray (LC-MS/MS), nous avons identifié les marqueurs de résistance en 1 à 2 heures. En 30 min, nous avons pu détecter les mécanismes de résistance aux β-lactamines, aux glycopeptides, à la méthicilline et aux fluoroquinolones, à l'aide de méthodes de type "Suivi de Réactions Sélectionnées", ou "Selected Reaction Monitoring" (SRM). Au cours de la même analyse multiplexée, des dizaines de protéines peuvent être détectées de façon hautement spécifique et sensible. Comme l'illustre l'étude de la résistance multifactorielle chez Acinetobacter baumannii, cette approche permet en outre une analyse quantitative d'un grand intérêt pour certains mécanismes de résistance. Cependant, malgré ces perspectives attrayantes, la LC-MS/MS reste, aujourd'hui, loin d'une possible implantation en routine dans les laboratoires de microbiologie. Les instruments sont trop coûteux et la technologie trop complexe pour un usage pour du diagnostic in vitro. La spectrométrie de masse pourrait déjà avantageusement compléter les technologies actuelles de biologie moléculaire. Aujourd'hui, le séquençage de nouvelle génération est la méthode de référence pour la caractérisation moléculaire des bactéries. Mais, comme démontré dans ce travail, l'annotation des gènes est perfectible. Pour quelques dizaines d'euros et quelques heures d'analyse, les peptides identifies par spectrométrie de masse facilitent l'assemblage des séquences (« scaffolds ») et la détection des gènes. De surcroît, la spectrométrie de masse permet une quantification précise des protéines. Elle apporte ainsi une nouvelle dimension analytique et une vision moléculaire plus proche du phénotype. En conclusion, la spectrométrie de masse LC-MS/MS peut être une technologie complémentaire attractive, voir une future alternative, à la biologie moléculaire pour la caractérisation des bactéries / Thanks to MALDI-TOF mass spectrometry, identification of isolated bacteria is now possible within a few minutes. But doctors also need to rapidly know the phenotype of resistance of the bacteria. Indeed, the patient mortality rate increases when the antibiotherapy is not appropriate. However, MALDI-TOF instruments are not able to analyze antibacterial resistance rapidly and comprehensively.Today, 6 to 24 hours are nedded for antibiotic susceptibility testing. When combining a liquid chromatography and a mass spectrometer with electrospray ionization (LC-MSMS), the detection of resistance biomarkers was possible within 1 to 2 hours. Using a Selected Reaction Monitoring (SRM) method, resistance mechanisms to beta-lactams, methicillin, glycopeptides and fluoroquinolones were detected in strains within 30 minutes. Tens of resistance determinants can be analyzed in a single multiplexed assay, with high specificity and sensitivity. Illustrated by the study of multifactorial resistance in Acinetobacter baumannii, the technology allows furthermore a quantitative analysis, which is of great value for some resistance mechanisms. Similarly, we identified virulent strains of enterohemorrhagic Escherichia coli by targeting toxins and serotype biomakers in the same assay. Mass spectrometry offered deeper bacterial characterization than conventional serotyping using polyclonal antibodies. However, despite all these favorable prospects, LC-MS/MS remains today far from reaching a routine use in microbiological hospital laboratories. Instruments are too expensive and the technology is too cumbersome for a daily in vitro diagnostic use. Waiting for a more suitable use, mass spectrometry could yet advantageously complement current molecular technologies. Today, the gold standard to study bacteria at molecular level is next generation sequencing. However, as demonstrated during this work, gene annotation remains imperfect. For tens of euros and few hours of analysis, peptides identified by mass spectrometry analysis of a bacteria might improve scaffold assembly and gene detection. Moreover, mass spectrometry gives an accurate protein quantitation and brings a new analytical dimension, potentially closer to the phenotype than molecular techniques. In conclusion, LC-MS/MS mass spectrometry could be an attractive complementary, or alternative technology in a near future, to conventional molecular biology techniques for deep characterization of bacteria

Capillary electrophoresis mass spectrometry (CE-MS) is a versatile instrumental method for metabolomics, which allows for comprehensive metabolite profiling of volume-limited biological specimens in order to better understand the molecular mechanisms associated with chronic diseases, including an alarming epidemic of obesity worldwide. Multiplexed CE separations enable high-throughput metabolite screening with quality assurance to prevent false discoveries when combined with rigorous method validation, robust experimental designs, complementary statistical methods, and high-resolution tandem mass spectrometry (MS/MS) for unknown metabolite identification. In this thesis, multiplexed CE-MS technology is applied for both targeted and untargeted metabolite profiling of various biological fluids, including covalently bound thiol-protein conjugates, as well as free circulating metabolites in serum and plasma, and excreted/bio-transformed compounds in urine due to complex host-gut microflora co-metabolism. This work was applied to characterize aberrant metabolic responses of obese subjects in response to dietary challenges, and measure the benefits of dietary interventions that reduce adiposity without deleterious muscle loss. Chapter 2 presents, a simple, sensitive yet robust analytical protocol to expand metabolome coverage in CE-MS for the discovery of labile protein thiols in human plasma using a rapid chemical derivatization method based on N-tert-butylmaleimide (NTBM). Chapter 3 describes targeted metabolite profiling of serum and plasma to investigate the differential metabolic responses between healthy and unhealthy obese individuals before and after consumption of a standardized high-caloric meal, respectively. Chapter 4 of this thesis describes an untargeted metabolite profiling strategy for urine using multisegment-injection (MSI)-CE-MS for elucidating the effects of protein supplementation following a short-term dietary weight-loss intervention study. This work revealed six urinary metabolites that were classified as top-ranking treatment response biomarkers useful for discriminating between subjects consuming carbohydrate (control), soy, and whey supplemented diets. In summary, this thesis demonstrated the successful implementation of multiplexed CE-MS technology for biomarker discovery in nutritional-based metabolomic studies as required for more effective treatment and prevention of obesity for innovations in public health. / Thesis / Doctor of Philosophy (PhD)

Biologische Diversität existiert sowohl zwischen mehreren Arten als auch innerhalb einer Art, innerhalb von Populationen und Individuen einer Population. Die intraspezifische Diversität wurde bislang ausgiebig auf der Ebene des Genoms untersucht. Sie ist im Kontext metabolischer Zusammenhänge in Pflanzen bisher kaum untersucht und es existieren nur wenige Veröffentlichungen zu diesem Thema. Uns sind bisher keine Publikationen zu Phytohormonen in Sesam bekannt. Neben dem wissenschaftlichen Interesse an der metabolischen Diversität in Sesam, spielen Stresshormone eine wichtige Rolle in der pflanzlichen Abwehr. Der Phytohormonspiegel im Samen ist unter agronomischen Gesichtspunkten relevant, da es vorkommen kann, dass Sesamsamen spontan auskeimen, während sie sich noch an der grünen Pflanze befinden. Diese Eigenschaft ist unerwünscht, da der wertvolle Samen auf diese Weise verloren geht. Im Rahmen dieser Arbeit wurde die Variation im Phytohormonniveau in 16 Akzessionen mit unterschiedlicher geographischer Herkunft untersucht. In Blättern und Wurzeln konnten ABA, JA, SA und SAG nachgewiesen werden, während GA4 lediglich in Blättern vorkam. Eine der Akzessionen aus Japan („Japan 2“) produzierte JA, SA und SAG in hohem Ausmaß. Hier konnten außerdem hohe Gehalte an Chitinasen festgestellt werden. Chitinasen sind für den Abbau von Chitin, dem Hauptbestandteil der pilzlichen Zellwand, verantwortlich. Eine Charakterisierung der Akzessionen mittels AFLP-Analyse zeigte, dass sich „Japan 2“ genetisch nicht mehr von anderen Akzessionen unterschied, als das Mittel der Unterschiede innerhalb aller gesammelten Proben. Bereits in früheren Untersuchungen unserer Arbeitsgruppe im Rahmen einer ungerichteten Metabolitenanalyse, konnte eine hohe Variabilität bei Sesamakzessionen gezeigt werden (Laurentin et al. 2006). Darüber hinaus, stimmen die Unterschiede im metabolischen Profil der Akzessionen nicht mit dem Grad ihrer genetischen Verwandtschaft überein. Es ist bekannt, dass tageszeitliche Unterschiede viele biologische Prozesse kontrollieren. Wir haben die tageszeitlichen Effekte auf den Phytohormonstatus untersucht und dabei die Unterschiede in Pflanzenorganen berücksichtigt. Tageszeitliche Konzentrationen von ABA, JA, IAA, SA und SAG wurden zu 8 unterschiedlichen Tageszeitpunkten in 3 unabhängigen Replikaten mittels HPLC untersucht. Wir konnten keine statistisch signifikanten Unterschiede erkennen. Die Untersuchungen zeigten jedoch eine Variation in den Phytohormonkonzentrationen in unterschiedlichen pflanzlichen Organen. Sekundäre pflanzliche Metabolite spielen als Resistenzfaktoren gegen Mikroorganismen eine wichtige Rolle. Sesamakzessionen, die diese Substanzen im hohen Ausmaß produzieren, stellen eine wichtige züchterische Ressource da. Um die Variation innerhalb der Akzessionen zu untersuchen, die ein hohes Niveau an sekundären Inhaltsstoffen aufweisen, haben wir die Effekte von 32 Pflanzenextrakten aus Sesamakzessionen gegen phytopathogene Pilze untersucht. Darunter befand sich ein Wurzelpathogen mit Spezialisierung auf Sesam (Macrophomina phaseolina), ein Blattpathogen mit breitem Wirtspflanzenkreis (Alternaria alternata) und Gefäßpathogen (Fusarium oxysporum). Die Diversität der Effekte, die für die unterschiedlichen Akzessionen beobachtet werden konnten, führt zu der Annahme, dass die Resistenzeigenschaften der Pflanzen durch gezielte züchterische Beeinflussung der metabolischen Aktivität verbessert werden können. In weiterführenden Untersuchungen zur Aufreinigung der Substanzen mit inhibitorischer Wirkung wurden Pflanzenextrakte in 80% Ethanol mit verschiedenen organischen Lösungsmitteln fraktioniert. Die meisten inhibitorischen Effekte konnten der Diethylether-Fraktion zugeschrieben werden. Im ersten Schritt wurden 4L des Extraktes hergestellt. Zwei aufgereinigte Lignane aus Sesam wurden gegen M. phaseolina, A. alternata und F. oxysporum getestet. Sesamin zeigte keinen Effekt bis zu einer Konzentration von 5mg/ml, während Sesamol (und 2,4-Dinitrophenol als Kontrolle) einen starken inhibitorischen Effekt aufwies. Für diese Substanzen wurden IC50 Werte ermittelt. Man kann festhalten, dass Sesamol dazu dienen kann, das Wachstum invasiver Pathogene einzuschränken. Durch die Kreuzung von zwei Elternlinien, die in der AFLP-Analyse einen signifikanten Polymorphismus aufwiesen, wurden Inzuchtlinien erzeugt. Die Nachkommen dieser Kreuzung wurden in 5 Generationen selbstbefruchtet. Das so entstandene Set aus RILs wurde mittels AFLP charakterisiert. Alle untersuchten RILs waren Hybride. Dies zeigt, dass während der ersten Kreuzung der Elternlinien keine Selbstung erfolgte. Wie erwartet, spalteten polymorphe AFLP-Marker der Elternlinien in den RILs zufällig auf. Monomorphe Marker fehlten in einigen RILs. Des Weiteren traten neue Marker auf, die zuvor nicht in den Elternlinien festgestellt werden konnten. Das Auftreten neuer Marker kann durch Rekombination zwischen Restriktionsfragmenten erklärt werden, welche die AFLP-Marker begrenzen. Die RILs werden nun von unseren Kooperationspartnern zum Aufbau einer genetischen Karte verwendet (Prof. Sami Doganlar und seine Arbeitsgruppe, Universität Antalya, Türkei).